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Flashcards in Proteomics Deck (30):
1

Define proteomics

The study of the entire complement of proteins, how they're modified, when and where they're expressed, how they're involved in metabolic pathways and how they interact with one another

2

What are proteins made up of?

Amino acids

3

What is special about proteins?

They all have distinctive molecular weights

4

Give an example of an enzymatic catalysis protein

p450

5

Give an example of a transport and storage protein

Haemoglobin

6

Give an example of a coordinated motion protein

Muscle proteins

7

Give an example of an immune protection protein

Antibodies

8

Give examples of proteins involved in the generation and transmission of nerve impulses

Rhodopsin and acetylcholine

9

Give an example of a protein involved in the control of growth and differentitation

Growth factor proteins

10

What are the two proteomics approaches?

Top down and bottom up

11

Briefly describe the bottom-up proteomics approach

1. Isolate proteins
2. Reduce and alkylate
3. Digest
4. Separate out peptides
5. Mass determine peptides and sequences
6. Informally collate all of the data to determine what the protein is and how much there is

12

Proteins are structurally complicated structures, true or false?

True

13

How can you break the four levels of protein structure?

By trypsin digest

14

Give an example of a reducing agent, how it works, and what the outcome is for proteins

Dithiothreitol (DTT)
Disrupts disulphide bonds to free thiol groups

15

Give an example of an alkylating agent, how it works, and what the outcome is for proteins

Iodoacetamide (IAA)
Maintains disrupted disulphide bonds by alkylating cysteine groups and thus preventing them from reforming disulphide bonds
Causes the formation of S-carbamidomethyl cysteine groups

16

What do digestive enzymes do?

Recognise specific amino acids and cleaves at this specific site only

17

What is the most commonly used digestive enzyme?

Trypsin

18

Where does trypsin cut?

At the C-terminus of lysine and arginine residues (except when either is followed by proline)

19

What sorts of peptides will be excluded from digestion analyses?

Very short and very long peptides

20

What do you need to do in order to maximise the liberation of tryptic peptides?

Need to disrupt the 3D structure of proteins

21

What is dithiothreitol (DTT) used for?

To reduce/disrupt disulphide bonds formed between cysteine groups

22

What is iodoacetamide (IAA) used for?

To alkylate cysteine groups to prevent the reformation of disulphide bonds

23

What is trypsin used for?

To generate MS amenable peptides

24

What do samples that are a mixture of peptides have a range of?

pI
Hydrophobicity
Size

25

What is utilised to establish a separation gradient on HPLC?

The wide range of hydrophobicities

26

As peptides elute from LC column what are they subject to?

Vaporisation, ionisation and detection by the mass spectrometer

27

What are ionised peptides subject to?

Tandem mass spectrometry to determine amino acid sequence

28

What does LC stand for?

Liquid chromatography

29

What is liquid chromatography similar to?

Paper chromatography

30

Liquid chromatography separates out molecules according to their what?

Hydrophobicity